A partially deficient and atypical equine transferrin variant, TF N

A new, partially deficient and phenotypically atypical transferrin variant, TF N, was detected in sera of a number of Finnhorses belonging to one family. The variant was inherited codominantly. In polyacrylamide gel electrophoresis (pH9.0) of sera, variant N appeared as a single weak band migrating slightly faster than the main anodal band of variant M. After immunoblotting or isolation an additional, still weaker, faster band was observed as well as some trace bands. The cathodal component, which is present in other transferrin variants, could not be convincingly proved. The main component of variant N contained four sialic acid residues.     

Nature of the heterogeneity within genetic variants of bovine serum transferrin

A comparison is made of the four main components of an homozygous variant (A or D₂, D₂) of bovine serum transferrin. These are designated I-IV in order of increasing mobility in electrophoresis at pH 7.5. Components I, II, HI and IV have 2,2,3 and 3 residues of sialic acid per transferrin molecule and appear to correspond to components 2a, 2b, 3a and 3b respectively of Stratil & Spooner (1971). The difference between components I and II and between III and IV does not reside in sialic acid differences. On the basis of peptide maps of reduced carboxami-domethylated components, urea-starch gel electrophoresis and quantitative sequence studies, it is concluded that components II and IV have a scission in the peptide chain. By homology with the sequence of MacGillivray et al. (1977) for human serum transferrin it is suggested that the scission occurs between residues 55 and 54 from the C-terminus and this portion of the chain has a 'molecular' weight of ca. 6000. The implications are briefly discussed.     

Studies on equine transferrin--I. The isolation and partial characterization of the D and R variants

Each of two genetic variants of equine transferrin, D and R, is isolated from the blood of the heterozygote by a gentle fractionation procedure at pH 7.2. It is shown by step gradient polyacrylamide gel electrophoresis at pH 7.9 that each of these phenotypes exhibits two major bands (designated F, fast, and S, slow) and several minor bands. Components corresponding to these bands are separated by ion-exchange chromatography at pH 6.6 and 6.9 respectively for the D and R variants. The F and S components of each variant contain respectively four and two sialic acid residues. The nature of their heterogeneity is, at least in part, due to their varying sialic acid contents. It has not been possible to desialylate them completely by neuraminidase. On the basis of comparative studies of the tryptic and chymotryptic peptide maps of transferrins D and R it is concluded that there are at least two amino acid substitutions--D:R:Asp:Gly and Glu:Gly. These two substitutions are qualitatively in accordance with the difference in the electrophoretic mobility between the two variants at alkaline pH.     

Partial characterization of horse transferrin heterogeneity with respect to the atypical type, Tf C

In starch gel electrophoresis of horse sera each transferrin variant is formed by a strong anodal band and a weaker cathodal band. An 'atypical' variant, Tf C, has two zones of about equal intensity. Family data show that Tf C is genetically controlled by an allele Tf C at the Tf locus. Frequencies of transferrin alleles in various horse breeds are also presented.
After isolation and fractionation of individual transferrin variants (Tf O, Tf D, Tf C) on DEAE-Sephad Summary ex, additional weak bands were detected. The two main zones of each variant were isolated in a pure state and treated with neuraminidase. In all three variants studied the electrophoretic mobility of the slower band (2a) was decreased in two steps, and the faster band (4b) in four steps. The mobilities of bands derived from the fast zone (4b) were slower than mobilities of corresponding bands derived from the slow zone (2a). These results suggest the presence of two sialic acid residues in the slow zone, and of four residues in the fast zone. Residual heterogeneity was independent of sialic acid.     

Studies on bovine transferrin; isolation and partial characterization

Bovine serum transferrin (type Tf-A) was isolated by a series of four techniques; (a) precipitation with Rivanol; (b) chromatography of the soluble protein fraction on a column of Sephadex G-150; (c) chromatography of the transferrin containing protein zone on a column of DEAE-Sephadex; and (d) chromatography on a column of DEAE-Sephadex after transferrin was treated with neuraminidase.
It was found that an unidentified protein binds firmly to transferrin, and its removal is only possible after the release of the sialic acid residues with neuraminidase. It is possible that this protein is hemopexin. The occurrence of multiple transferrin components is, in part, dependent on the number of sialic acid residues; possible differences in molecular weight or size seem not to be a factor. The amino acid composition of bovine transferrin, and that of each of three subfractions, resembles that of human transferrin. The calculated mol. wt. of bovine transferrin was found to be 67,000 from sedimentation and viscosity data and 72,400 from sedimentation and diffusion measurements. Sedimentation and viscosity data in concentrated urea suggest that bovine transferrin is composed of two subunits, an observation which is in contrast to data from studies which suggest that human transferrin is composed of a single polypeptide chain.     

Evidence for transferrin polymorphism in Spanish wild rabbits

Serum samples from 412 Spanish wild rabbits were analysed by starch and polyacrylamide gel electrophoresis. Three different transferrin (Tf) phenotypes (A, AB and B) were observed by both methods. The occurrence of two codominant alleles (TfA and TfB with frequencies of 0.89 and 0.11 respectively) at an autosomal locus (Tf) was supported by the population data on genetic equilibrium. Electrophoretic mobility differences between the Tf variants A and B could not be explained by differences in sialic acid or iron contents. Each of the two Tf variants were shown to have two sialic acid residues by neuraminidase treatment. These variants had similar affinities for iron, and iron binding did not lead to the conversion of one variant into the other.     

Isolation and properties of individual components of cattle transferrin: The role of sialic acid

Homozygous cattle transferrin has been fractionated into six main peaks by DEAE-Sephadex chromatography. These correspond to the six transferrin components seen in starch gel electrophoresis of normal serum. In addition, six more minor components were isolated from DEAE-Sephadex, making 12 in all, and these could be divided into six pairs. Treatment of whole transferrin with neuraminidase yielded only two bands. Treatment of the individual fractionated bands showed that the slower band of each pair had the same mobility as the slow band from treated whole transferrin, while the faster band from each pair corresponded with the fast band of treated whole transferrin. These observations, and the results of sialic acid assays, showed that the difference between the pairs of bands was caused by differing numbers of sialic acid residues (0–5) per molecule of protein, but that sialic acid was not responsible for the difference between the bands within a pair. The mode of genetic control is discussed and probably involves three loci. Other physicochemical properties of cattle transferrins, namely, molecular weight, effect of iron addition, and behavior in isoelectric focusing, were also studied.     

Molecular weight heterogeneity of bovine serum transferrin

Cattle transferrin (Tf) was purified from serum of variant A and four bands were isolated. The peptide patterns of these bands when cleaved by proteases and by cyanogen bromide (BrCN) were compared, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Variant A displays two groups of molecules--large (L) and small (S)--on SDS-PAGE; the molecular weight of the L bands is 78,400 +/- 1700 and that of the S bands is 72,000 +/- 1700. However, S-band molecules could not be produced artificially by heat treatment of L bands in the presence of SDS and 2-mercaptoethanol. Since deglycosylated Tf also showed molecular weight heterogeneity, the sugar moieties of Tf other than sialic acids were not the cause of the heterogeneity. These results suggest that heterogeneity within a given variant is due to the presence of two kinds of molecule of different molecular weight. The peptide patterns of L and S bands produced by proteases and those produced by BrCN were distinctly different from each other. However, the stepwise degradation patterns of L and S bands resembled each other when treated with both chymotrypsin and BrCN. This suggests that L-band molecules differ from S-band molecules only in the presence of an additional carboxyl-terminal peptide.     

Polymorphism of transferrin in carp (Cyprinus carpio L.): Genetic determination,isolation, and partial characterization

Seven transferrin variants (A, B, C, D, E, F, and G) have been found in carp sera (Cyprinus carpio L.). Genetic analysis involves five variants and agrees with the hypothesis of simple codominant autosomal inheritance at one transferrin (Tf) locus in spite of the fact that the carp is a tetraploid in relation to other species of the same family. Carp populations from three regions were studied which differed in gene frequencies. Individual populations were in Hardy—Weinberg equilibrium. The polymorphism of carp transferrins can be used for the identification of offspring of single parent pairs, stocked in one pond. Transferrins have been isolated and characterized. Homozygous phenotypes comprised four iron-binding components differing in electrophoretic mobility. This heterogeneity is not caused by sialic acid, which is absent. Amino acid composition, content of hexoses (1 mole/mole of protein) and hexosamines (1 mole/mole of protein), molecular weight (70,000), and the isoelectric point (5.0) have been determined. No N-terminal amino acid could be detected.     

Evidence of homology in a high-sulphur protein fraction (SCMK-B2) of wool and hair α-keratins

Fractions corresponding to the S-carboxymethylated high-sulphur protein component SCMK-B2 isolated by Gillespie (1963) from Merino wool were prepared from five different wool samples and also from bovine hair. The six fractions showed great similarities in amino acid composition, and also gave very similar peptide ;maps' after tryptic and chymotryptic digestion. Some of the peptides were isolated from the different samples, and evidence is given that suggests that a sequence of at least 21 amino acids is common to all the fraction SCMK-B2 preparations. Further, all the fractions derived from the wool samples have the same acetylated heptapeptide for the N-terminal sequence, but one extra residue may be present in this N-terminal sequence in the protein from bovine hair. The general significance of these findings is discussed.     

Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

Separation of both the Bbeta- and the gamma-polypeptide chains of human fibrinogen into two main types which differ in sialic acid content

The gamma- and Bbeta-polypeptide chains of purified human fibrinogen have each been resolved into two major species: gammaL and gammaR and BbetaL and BbetaR. These molecular variants, separable on CM-cellulose, differ from each other in sialic acid content: approximately 2 residues of sialic acid per molecule of polypeptide chain for the L species to 1 residue of sialic acid per molecule for the R species. The two types of each polypeptide are demonstrable in preparations of fibrinogen from single donors as well as in pooled fibrinogen. The L and R forms of the gamma chains or the Bbeta chains do not differ in their electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting that they are similar in molecular weight. They are also indistinguishable in polyacrylamide gels in the presence of urea at pH 2.7. Maps of ninhydrin-positive tryptic peptides of the L and R forms of the gamma chain displayed differences within a small group of peptides which have been shown to contain the sialic acid residues present in the gamma-polypeptides. No differences between the peptide maps of BbetaL and BbetaR chains were obvious. A larger ratio of L/R in the gamma and Bbeta chains of dysfibrinogenemia fibrinogen "Zürich II" than in those of normal fibrinogen explains the higher content of sialic acid measured in the native Zürich II fibrinogen molecule.     

Structural studies on rabbit transferrin: isolation and characterization of the glycopeptides

The structure of rabbit transferrin was investigated with regard to number, size, and composition of the heteropolysaccharide units and their relative location on the polypeptide chain. The composition and molecular weight of the Pronase glycopeptides revealed that rabbit transferrin contains two heteropolysaccharide units, each composed of 2 sialic acid residues, 2 galactose residues, 3 mannose residues, and 4-N-acetylglucosamine residues. The composition and molecular weight of the tryptic glycopeptides further substantiated the existence of two identical heteropolysaccharide units and revealed that both units have identical amino acid residues in the immediate vicinity of the carbohydrate attachment sites to the polypeptide chain, suggesting a sequence homology surrounding the two glycosylation sites. Characterization of the cyanogen bromide fragments from rabbit transferrin indicated that both heteropolysaccharide units are located within a single polypeptide fragment representing approximately one-third of the molecule.     

Comparison of bovine serum transferrin A and D2. II. Glycopeptides

Glycopeptides are isolated from subtilisin and pronase digests of whole bovine serum transferrin A and D2. The two variants yield glycopeptides with identical amino acid composition. Hence, there is probably no amino acid substitution in this region of the peptide chain. Amino acid sequence determination of one glycopeptide (subtilisin glycopeptide 8) gives the sequence: (CHO)Asn-Ser-Ser-Leu-Cys. This sequence is identical with that of residues 491-495 of the sequence for human serum transferrin (MacGillivray et al., 1982) except that in the bovine transferrin, Asp is replaced by Asn, enabling carbohydrate attachment. A second glycopeptide sequence Arg-(CHO)Asn-Ala-Thr-Tyr is observed, and the significance discussed in relation to carbohydrate moieties of serum glycoproteins.     

Comparison of casein of cynomolgus monkey (Macaca fascicularis) with human casein

Casein of cynomolgus monkey was compared with those from human and bovine milk. Cynomolgus monkey casein showed similar electrophoretical patterns to those of human casein on Disc- and SDS-electrophoresis. It consisted of beta- and kappa-casein-like components. The component corresponding to bovine alpha s1-casein was not detected. The beta-casein-like fraction of cynomolgus monkey showed 9 bands on Disc-PAGE. These were suggested to be the same protein binding different levels of phosphorus by dephosphorylation experiment using an acid phosphatase. The kappa-casein-like component of cynomolgus monkey was highly glycosylated (about 50% carbohydrate) similarly as human kappa-casein and the constituent carbohydrates were same as those detected in human kappa-casein (galactose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid). Amino acid composition of cynomolgus monkey kappa-casein bore a resemblance to those of both human and bovine kappa-caseins. Amino acid composition of cynomolgus monkey beta-casein was also similar to those of human and bovine beta-caseins.     

Comparison of bovine serum transferrin A and D2. II. Glycopeptides

Glycopeptides are isolated from subtilisin and pronase digests of whole bovine serum transferrin A and D₂. The two variants yield glycopeptides with identical ami-no acid composition. Hence, there is probably no amino acid substitution in this region of the peptide chain. Amino acid sequence determination of one glycopeptide (subtilisin glycopeptide 8) gives the sequence: (CHO)Asn-Ser-Ser-Leu-Cys. This sequence is identical with that of residues 491–495 of the sequence for human serum transferrin (MacGillivray et al., 1982) except that in the bovine transferrin, Asp is replaced by Asn, enabling carbohydrate attachment. A second glycopeptide sequence Arg-(CHO)Asn-Ala-Thr-Tyr is observed, and the significance discussed in relation to carbohydrate moieties of serum glycoproteins.     

Polymorphic variation in the amount of sialic acid attached to bovine transferrin

In this paper family studies are presented which support the hypothesis of polymorphism in the process controlling sialic acid binding to bovine transferrin which modifies its phenotype as seen in starch gel electrophoresis. It has been shown that this polymorphism is controlled by a locus Tfs with two alleles Tfs A and Tfs a. Tfs a/a animals have the abnormal phenotype with the two faster bands of the four bands of a normal transferrin allele being virtually absent. Tfs A/a and Tfs A/A are phenotypically normal. Limited evidence is presented which suggests that the Tf and Tfs loci are not linked.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

Iron uptake from sialo transferrins by rat reticulocytes

Preparative isoelectric focusing of human diferric transferrin preparations yielded seven bands with different isoelectric points, due to differences in sialic acid content. Incubation of rat reticulocytes at 37 and 4 degrees C with differic preparations of four of these transferrin forms labeled with 59Fe and 125I show no differences in membrane binding of iron and transferrin and in iron uptake. Hence it is concluded that the carbohydrate chains are not directly involved in the process of iron delivery to reticulocytes.     

BoLA class I charge heterogeneity reflects the expression of more than two loci

Internationally recognized allo-antisera in lymphocyte microcytotoxicity assays are thought to detect allelic products of a single highly polymorphic class I locus. A recent report suggested that two bovine lymphocyte antigen (BoLA) class I loci are expressed at the protein level. However, 1D-IEF analysis of BoLA class I molecules reveals multi-band patterns which cannot be reconciled with the reported number of loci. The aim of this study was to investigate the origins of the charge diversity of BoLA class I molecules observed using 1D-IEF. BoLA class I molecules appear to be glycosy-lated at a single N-linked position with a complex type carbohydrate moiety which has up to three terminal sialic acid residues. Class I molecules immunoprecipitated from resting bovine PBL are not phosphorylated. Neither modification is responsible for the observed charge heterogeneity. Peptide mapping reveals that different BoLA charge variants have distinct digestion patterns. Furthermore, a number of different polypeptides are associated with each serological specificity. These polypeptides appear to be encoded by different loci which exist in linkage disequilibrium. The number of charge variants with different peptide maps indicates that the BoLA system has a minimum of three class I loci expressed at the protein level.